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A common feature of cytokine receptor activation is ligand-induced receptor aggregation involving the homo- or hetero-dimerization of two or more receptor components and their assembly into a fully functional signaling complex. Structural data for hetero-dimeric receptor families that utilize a common binding and signaling subunit such as the IL-2 and IL-6 receptor systems have provided unique insights into their functional activation (Wang et al, Science 310:1159, 2005; Stauber et al, Proc. Natl. Acad. Sci. USA 103:2788, 2006; Boulanger et al, Science 300:2101, 2003). The GM-CSF, IL-3, and IL-5 family of receptors remains the last major group of class I hematopoietic receptor systems to be structurally elucidated.
The GM-CSF, IL-3 and IL-5 family of cytokines regulates the survival, proliferation, differentiation and functional activation of hematopoietic cells with GM-CSF also controlling dendritic cell and T cell function, thus bridging innate and acquired immunity (Guthridge et al, Stem Cells 16:301, 1998). Accordingly, GM-CSF is used for the expansion of hematopoietic cells after bone marrow transplantation, for the activation of mature cell function in infectious diseases, and as an adjuvant to bolster anti-tumor immunity (Fleetwood et al, Crit. Rev. Immunol. 25:405, 2005). Conversely, GM-CSF, IL-3 and IL-5 have all been implicated in multiple pathologies resulting from excessive or aberrant expression of the cytokines or their receptors, in conditions such as arthritis, asthma, autoimmunity and leukaemia (Guthridge et al, supra 1998). Their receptors are expressed at very low level (100-300 per cell) on the surface of hematopoietic cells and comprise a cytokine-specific alpha subunit and a beta subunit (βc) that is common to all three receptors (Guthridge et al, supra 1998). Each alpha subunit binds cytokine with low affinity (0.2 nM to 100 nM) but the presence of βc converts this to high affinity (100 pM) causing dimerization of both subunits and receptor activation. Structure-function studies of GM-CSF, IL-3 and IL-5 and their receptors have noted regions of importance for ligand binding and receptor activation while receptor mutants that can function in a ligand-independent manner have suggested novel activation mechanisms. However, the structural elements and underlying mechanisms that can explain both forms of receptor activation remain elusive.
Activation of the GM-CSF receptor family by ligand-induced receptor dimerization, shows similarities and important differences to the IL-2 and IL-6 systems. First, in the GM-CSF receptor family, the alpha and beta subunits both participate in signaling such that deletion of the alpha chain cytoplasmic domains abolishes receptor function, suggesting an essential membrane proximal interaction of both subunits is required for receptor activation (Lia, J. Biol. Chem. 271:28287, 1996). Secondly, some of the GM-CSF receptor exists as a preformed complex, a finding that explains the rapid association kinetics of GM-CSF and the ability of the GM-CSF receptor to be co-immunoprecipitated with IL-3 and IL-5 receptors following stimulation with IL-3 or IL-5, respectively (Woodcock et al, Blood 90:3005, 1997). Thirdly, specific mutations in the membrane proximal region of βc induce ligand-independent signaling yet depend on co-expression of the alpha subunit (D'Andrea et al, Blood 87:2641, 1996; Jenkins et al, J. Biol. Chem. 271:29707, 1996). Finally, the existence of βc, either on the cell membrane or in solution, as a non covalently-linked dimer is puzzling, particularly as the crystal structure of isolated βc revealed a dimer in which the cytoplasmic tails were 120 Å apart (Can et al, cell 104:291, 2001). It is difficult to envisage how such a distance would allow transphosphorylation of βc by their associated JAK-2 kinases and downstream signaling.
There is a need to determine the structure of the GM-CSF and GM-CSFR complex and its role in signaling in order to rationally design therapeutic agents useful inter alia in treating inflammation, cardiovascular disease, ischemia, brain and heart infarcts, aberrant immunity, cancer including leukemia and infection by pathogenic agents.